Fetal alcohol syndrome is an important cause of mental retardation. Exposure of the developing brain to alcohol can induce neuronal death, which contributes strongly to the learning deficits and neurological problems associated with FAS. Understanding the factors that influence neuronal vulnerability to alcohol-induced cell death in the developing brain is of considerable importance. We hypothesize that this maturation-dependent alcohol resistance is due to the acquisition of an inherent neuroprotective signaling pathway mediated by VPAC1, a G-protein-coupled receptor. This proposal will elucidate the function and molecular mechanisms of the VPAC1 signaling pathway, including one of its key downstream effector molecules, cAMP-Responsive Element Binding protein (CREB), in protecting developing neurons against alcohol-induced death. The first specific aim will assess the protective effect of the VPAC1 signaling pathway against alcohol toxicity in vitro and in vivo. We will trace the developmental expression of the pathway components and their downstream target neuroprotective genes and will determine whether the ontogeny of these genes coincides with the acquisition of time-dependent alcohol resistance. We will compare the seventy of alcohol-induced neuronal loss in Purkinje and granule cell cultures derived from mice with and without a functional VPAC1 receptor and will determine whether neurons lacking this receptor can be "rescued' from alcohol toxicity by the targeted delivery of VPAC1 carried by gene therapy vectors. We will determine whether the ectopic expression of VPAC 1 during the vulnerable period can prevent alcohol-induced death in cerebellar neurons. The second specific aim will explore the molecular mechanism by which the VPAC1 signaling pathway leads to neuronal protection. We will selectively silence VPAC1 and CREB using siRNA and shRNA technology and will assess the effect of this gene silencing on neuronal survival. We will determine whether specific silencing of VPAC1 or CREB can abolish the resistance of cerebellar granule and Purkinje neurons against alcohol-induced cell death during the resistant phase. We will determine whether silencing of VPAC1 or CREB affects the expression of neuroprotective and anti-apoptolic genes carrying Camp response elements in their promoters. These studies will provide vital information regarding the role and mechanism of an important G-protein-mediated signaling pathway in neuroprotection against alcohol. The reagents and results derived from these in vitro experiments will lay the foundation for the gene therapy studies planned for in vivo applications in the future.